Plants utilize nitrogen to form organic compounds. Ammonia and ammonium ions do not accumulate in plant cells but instead are rapidly assimilated. Ammonium assimilates through two possible pathways. The first pathway produces glutamate and is catalyzed by glutamate dehydrogenase (GDH), which is found in chloroplasts and mitochondria.
The second pathway for assimilation of ammonia involves a reaction with glutamate to form its amide, glutamine. This reaction is catalyzed by glutamine synthase (GS) and requires energy in the form of ATP. Glutamine is then catalyzed by glutamate synthase (GOGAT) to form glutamate. GS appears in chloroplasts and cytosol in leaves and roots, whereas, GOGAT is in leaf chloroplasts and plastids in roots.
Although both pathways result in glutamate, the second pathway appears more important in ammonium assimilation in plants. Glutamate dehydrogenase, the enzyme of the first pathway, has a high Km value. This value which is the concentration of ammonia where half of the enzyme maximum operation rate is within levels which are toxic for plant cells. In contrast, the GS Km value is much lower. Additionally, radioactive labeling of NO.sub.3 or NH.sub.4 show labeled nitrogen in the amide group of glutamine first.
Although GS has a high affinity for ammonia and GDH has a lower affinity, GS has low specific activity per enzyme molecule and GDH has high specific activity per molecule.
Ammonium assimilation pathways of plants and microorganism; although maybe not fully understood; have been known. In October of 1980, the ICI Agricultural Division published in Nature, Volume 287, page 396 an article on improved conversion of methanol to single cell protein by Methylophilus methylotropus.
The researchers cloned the glutamine dehydrogenase gene of Escherichia coli (E. coli) into a mutant of Methylophilus methylotropus organism that lacks GOGAT. The paper explained that the GDH pathway should result in the organism consuming less energy. The researchers speculate that potential industrial or agricultural savings could be made by identification of features that incur "energy penalty" and this is an exciting area for recombinant DNA. This organism to organism transfer of the E. coli GDH gene should substantially decrease in enzyme activity thus a plasmid with a high copy number was used.
In 1988, the expression of E. coli glutamate dehydrogenase in cyanobacterium was reported in Plant Molecular Biology, Volume II, pages 335-344. Cyanobacterium that lacked glutamate dehydrogenase were transformed with the gdhA gene of E. coli and levels of NADP-specific glutamate dehydrogenase activity resulted in the transformed microorganism. The authors speculate that it would be interesting to investigate the engineering of glutamate dehydrogenase activity to higher plants and to study in detail the possible roles for glutamate dehydrogenase activity in ammonium detoxification.
Although there was some speculation on nitrogen assimilation genes in higher plants, in a paper on nitrogen assimilatory genes in The Genetic Manipulation of Plants and its Application to Agriculture, at page 109, the authors state that it would be tempting to suggest that crop plants might show increased metabolic efficiency if ammonium assimilation was channeled through glutamate dehydrogenase. But the authors clearly list the number of technological barriers to this. There remained a number of barriers to this research including the potential negative consequences of uncontrolled expression in the plant. The authors reluctantly conclude "perhaps" there may be some benefit in replacing glutamate synthase, with ammonium--utilizing alternatives.
In Molecular and General Genetics in 1993 in volume 236, pages 315-325, the modulation of glutamine synthetase gene expression in tobacco was reported. An alfalfa gene was placed in the tobacco plant cells in the sense and antisense position. Partial inhibitation in the antisense position was seen without a true homologous gene.
In 1994, it was reported that increasing the activity of plant nitrogen metabolism enzymes may alter plant growth, development and composition. Increased yield and protein content as well as reduced levels of nitrogen in agricultural runoff water and food may result. Plant nitrogen metabolism has been altered by transformation with a highly active assimilatory bacterial glutamate dehydrogenase gene, plant glutamate dehydrogenase is less active in ammonium gene has been altered by PCR and PCR strand overlap exchange to modify coding region and allow high levels of expression in plant cells. The 5' non-coding region has been altered to increase translation and permit protein targeting to either cytosol or chloroplasts. The 3' non-coding region has been altered to stabilize the mRNA and ensure appropriate polyadenylation of the mRNA. Certain codons likely to inhibit expression to high levels in plant cells have been altered. The effects of the various sequence substitutions on gene expression in plant cells compared to the unmodified gene will be reported. This abstract is reporting on speculation of the researchers as the abstract clearly reference what may happen or codons that are likely to inhibit. The abstract appears to provide a guess as to what might happen, not something that has been done.
Although researchers speculated that the gdhA gene may be useful in higher plants, the drawbacks and possible disruption of the photosynthesis pathway lead researchers to the belief that the potential use was probably not possible due to technical barriers. Even the inventor was only speculating on the potential of the gdhA gene to avoid ammonia toxification.
There remains a need to transform cereals to determine if the gdhA gene would have any effect on the plant in either nontoxifying levels or toxic levels of ammonia. The usefulness of the gene as a tolerance mechanism for certain herbicides was not proven prior to this. This gene is tolerant to phosphinothricin which can include glufosinate and other similar herbicidally active derivatives, salts, and acids thereof. The combination of this gdhA gene with other selectable markers to increase plant resistance to herbicide damage was heretofore undiscovered. The ability of a plant to increase dry weight due to increased nitrogen uptake in even nontoxic levels of ammonia was not realized or considered until the present invention.
The composition of proteins, sugars, starch, cellulose and structural lipids, storage lipids and oils can be altered by increasing or decreasing nitrogen supply to the plant. The question remains if the supply of nitrogen is at a high level can the composition of proteins, sugars, starch, cellulose, lipids and oils be modified by the addition of the gdhA gene. The present invention clearly indicates that the protein content in seeds and leaves is altered. Although the gdhA gene may have had some suggested potential to assimilate additional nitrogen in highly toxic nitrogen conditions, the gdhA genes result GDH enzyme has a weaker ammonium affinity than the ATP specific GS. At lower ammonium concentrations assimilation by GDH was expected to be limited due to its lower ammonium affinity and the reversibility of its reaction. Thus, it was surprising and unexpected that the gdhA gene when in a plant produced measurable changes in the number of leaves and protein content of the leaves and the seeds, the dry weight of the plant even in soils having normal ammonium levels. At these levels, the expectation would be that the GS/GOGAT cycle would be the active cycle.